A cross-sectional study was conducted in Portuguese schoolchildren and adolescents aged 6–18 years recruited from Agrupamento de Escolas de Montelongo and Associação Cultural e Recreativa de Fornelos, both located in the municipality of Fafe, northern Portugal, with all examinations performed in a room specially adapted for these measurements. Inclusion criteria were: best-corrected visual acuity (BCVA) ≥20/25 in both eyes, absence of ocular pathology, no history of ocular surgery, and no systemic disease known to affect ocular structure. The study was approved by the Ethics Committee for Research in Life and Health Sciences of the University of Minho (CEICVS n° 179/2024) and adhered to the tenets of the Declaration of Helsinki. Written informed consent was obtained from parents or legal guardians, and assent was obtained from children when appropriate. A total of 605 children were included: 127 myopes, 263 emmetropes, and 215 hyperopes.

Participants were classified into three refractive groups based on non-cycloplegic refraction: Myopia: spherical equivalent (SE) ≤ −0.50 D; Emmetropia: −0.50 D < SE < +0.50 D and Hyperopia: SE ≥ +0.50 D18,19

Exclusion criteria included best corrected visual acuity worse than 0.25 logMAR, anisometropia, previous ocular surgery, ocular pathology, or systemic conditions known to affect ocular structures. To avoid inter-eye correlation, only the right eye of each participant was included in the analysis.

Monocular visual acuity was measured using an ETDRS chart at a distance of 4 m and recorded in logMAR units. Objective non-cycloplegic refraction was obtained using a Plusoptix A16 autorefractor (GmbH, in Nuremberg, Germany). Cycloplegia was not performed in the present study due to several factors: (1) practical limitations associated with conducting large-scale school-based screenings under restricted medical supervision; (2) the intention to maintain methodological consistency with previous large epidemiological studies in pediatric myopia that used non-cycloplegic refraction together with suitable refractive error cut-offs20; and (3) the need to minimize possible side effects and parental concerns related to the use of cycloplegic drugs in a community-based setting. Nevertheless, we recognize that non-cycloplegic refraction can lead to an overestimation of myopia prevalence because of residual accommodation, especially among younger children with higher accommodative capacity. The extent of this effect may differ according to age and baseline refractive status, potentially influencing associations involving age-dependent factors such as educational level and academic achievement.18 Additional subgroup analyses restricted to participants aged ≥12 years were performed to evaluate the potential influence of accommodative bias associated with non-cycloplegic refraction.

Axial length (AL) and corneal curvature (keratometry) was measured using optical biometry (IOLMaster, Carl Zeiss Meditec). Refraction (sphere, negative-cylinder power, and axis) was converted into power vector components M, J0, and J45 using the Thibos method.21

All OCT imaging was performed using the Mocean 3000 (SLOOCT; MOPTIM), a combined Scanning Laser Ophthalmoscope and OCT platform that provides high-resolution retinal imaging with simultaneous SLO fundus registration, enabling precise automated layer segmentation of retinal and choroidal structures using spectral-domain OCT technology. All OCT examinations were performed in the morning, between 10:00 and 12:00 h, to minimise the influence of diurnal variation on choroidal thickness measurements, consistent with published recommendations.22,23 Only right eyes were included in the analysis. Scans with signal strength below 7/10, motion artefacts, or poor segmentation (signal <8/10) were excluded.

Macular thickness and volume were assessed using a macular cube scan protocol. ILM-RPE total retinal thickness and volume were automatically segmented by the device software. Subfoveal choroidal thickness (SFCT) was measured automatically by the Mocean 3000 software using automated segmentation algorithms that delineate the retinal pigment epithelium (RPE) and the choroid-scleral interface, without manual caliper measurement. The Mocean 3000 SLOOCT platform does not use Enhanced Depth Imaging (EDI) mode; choroidal visualisation was achieved through the standard SLOOCT acquisition protocol, which provides adequate depth penetration for reliable choroidal boundary detection. Peripapillary RNFL thickness was measured using an optic disc cube scan. Global RNFL thickness and quadrant-specific values (superior, inferior, nasal, temporal) were automatically calculated. Optic disc area and cup-to-disc ratio were also recorded from the same scan.

Statistical analyses were performed using SPSS (version 26, IBM Corp.). Continuous variables were expressed as mean ± standard deviation (SD). Differences between refractive groups were analysed using one-way ANOVA for normally distributed variables or the Kruskal–Wallis test for non-normal distributions. Post-hoc pairwise comparisons used Bonferroni corrections when applicable. Associations between axial length and OCT parameters were assessed using Pearson or Spearman correlation coefficients. To account for potential confounding factors, multivariable linear regression models were constructed for the primary OCT outcomes, including retinal nerve fiber layer (RNFL) thickness, choroidal thickness, and foveal thickness, as dependent variables. Age, sex, axial length, and refractive group were entered simultaneously as independent variables. Regression coefficients (B), standardized beta coefficients (β), 95% confidence intervals (95% CI), and adjusted coefficients of determination (adjusted R²) were reported. Multicollinearity was assessed using variance inflation factors (VIF), with no evidence of relevant collinearity observed. In addition, age-stratified normative reference values were generated for clinically relevant age categories (6–9, 10–13, and 14–18 years). For each group, the 5th, 50th, and 95th percentiles were calculated for the main OCT parameters. Statistical significance was set at p < 0.05.

Foveal and central macular thickness (Fovea thickness and Central thickness) did not vary significantly across groups (p > 0.30, Table 2). In contrast, both total macular area and macular volume were slightly lower in myopes compared with emmetropes and hyperopes, with statistically significant differences (p = 0.033 and p = 0.034). Most ILM-RPE thickness parameters showed no significant group differences; however, the superior-superior, temporal-temporal, and inferior-inferior sectors presented significantly thinner values in myopes (p = 0.003, p = 0.025, and p = 0.001, respectively, Fig. 1). Similarly, volumetric ILM-RPE measures were largely comparable across groups (Fig. 2), except for the superior-superior, temporal-temporal, and inferior-inferior sectors, where myopes exhibited significantly lower volumes (p = 0.003, p = 0.026, and p < 0.001).

Fig. 1.

Macular retinal thickness in myopic (A), emmetropic (B), and hyperopic (C) eyes. The post-hoc statistical significance between the three refractive groups is shown in yellow (p < 0.050).

Fig. 2.

Macular retinal volume in myopic (A), emmetropic (B), and hyperopic (C) eyes. The post-hoc statistical significance between the three refractive groups is shown in yellow (p < 0.050).

Significant differences were observed among refractive groups for several peripapillary and optic nerve head metrics (Table 3 and Fig. 3). Myopes showed thinner global RNFL (108.29 ± 11.08 µm) compared with emmetropes (114.18 ± 11.82 µm) and hyperopes (112.54 ± 11.18 µm; p < 0.001). Optic disc area was also smaller in myopes (p = 0.003), and both mean and vertical cup-to-disc ratios were significantly lower in this group (p = 0.001 and p = 0.002, respectively). Cup volume differed modestly across groups (p = 0.036).

Fig. 3.

RNFL thickness at quadrants and clock hours for myopes (A), emmetropes (B) and hyperopes (C) eyes. The post-hoc statistical significance between the three refractive groups is shown in yellow (p < 0.050).

Sectoral RNFL analysis showed consistently thinner values in myopes across most regions, with significant differences in nasal, superior, temporal, and inferior quadrants (p < 0.05 for all). Hour-wise RNFL measurements revealed a similar pattern, with myopes presenting significantly reduced thickness at multiple clock-hours, including 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 9 h, and 12 h (all p < 0.05). No significant differences were observed at 7 h, 8 h, 10 h, or 11 h. The Bonferroni post-hoc analysis shows that in the nasal part (from 12 o'clock to 5 o'clock), myopic patients have a lower RNFL (between 6 and 10%) when compared to emmetropic and hyperopic patients (p < 0.05).

Central choroidal thickness differed significantly among refractive groups (p = 0.001). Emmetropic children exhibited the greatest mean choroidal thickness (316.58 ± 51.46 µm), whereas thinner values were observed in hyperopic (298.70 ± 52.46 µm) and myopic participants (298.55 ± 58.52 µm). Bonferroni post hoc analysis revealed statistically significant differences between emmetropes and myopes (p = 0.009), as well as between emmetropes and hyperopes (p = 0.002). No significant difference was observed between myopic and hyperopic groups (p = 1.000). A weak but statistically significant negative correlation was identified between axial length and central choroidal thickness (r = −0.181, p < 0.001, Fig. 4), indicating progressive choroidal thinning with increasing axial length.

Fig. 4.

Correlation between axial length and central choroidal thickness.

Additional multivariate linear regression analyses were performed for the main OCT parameters, adjusting for age, sex, axial length, and refractive group (Table 4). Optic disc area was additionally included in the RNFL model. For global RNFL thickness, larger optic disc area (B = 8.93, p < 0.001) and shorter axial length (B = −2.37, p < 0.001) were independently associated with thicker RNFL values, whereas refractive group was no longer significant after adjustment (p = 0.813). For choroidal thickness, older age (B = 3.00, p < 0.001), shorter axial length (B = −18.21, p < 0.001), and refractive group (B = −8.42, p = 0.012) remained significant independent predictors. For foveal thickness, only axial length remained significantly associated (B = 3.14, p = 0.018), while age, sex, and refractive group were not significant.

Age-stratified normative percentile ranges were generated for the main OCT outcomes (Table S1). RNFL thickness remained relatively stable across age groups, with median values ranging from 110.7 to 114.1 µm. Choroidal thickness showed a modest increase with age, with median values rising from 297 µm in children aged 6–9 years to 315 µm in adolescents aged 14–18 years. Foveal thickness showed limited age-related variation, with median values around 212–216 µm across all age strata.

Our findings extend and complement prior Portuguese pediatric OCT studies. Queirós et al. (2015) reported mean global RNFL of 97.90 µm in 153 Portuguese children aged 4–17 years using Cirrus HD-OCT,14 while Gama et al. (2018) found pRNFL mean 100.19 ± 10.10 µm in 358 Portuguese children (mean age 6.41 years) using the same device.15 In comparison, our emmetropic cohort showed higher mean global RNFL values (114.18 ± 11.82 µm), with myopes presenting 108.29 ± 11.08 µm and hyperopes 112.54 ± 11.18 µm.

These differences likely reflect inter-device variability in segmentation algorithms between the Mocean 3000 (SLOOCT) used in the present study and the Cirrus HD-OCT used in prior Portuguese studies. The Mocean 3000 is a combined SLOOCT platform that differs from spectral-domain systems such as the Cirrus HD-OCT in its acquisition and segmentation approach; direct numerical comparison of absolute RNFL values between studies using different devices should therefore be made with caution. Differences in age range, sample composition, and measurement protocols may also contribute. Our SFCT data represent the first reported choroidal thickness measurements in a Portuguese paediatric cohort, filling a gap in the existing normative literature.

Our findings also align with and extend data from comparable-ethnicity European populations. Barrio-Barrio et al. (2013) reported mean global RNFL of 97.40 ± 9.0 µm and central macular thickness 253.85 ± 19.76 µm in 283 Spanish Caucasian children aged 4–17 years using Cirrus OCT.16 Al-Haddad et al. (2014) provided normative RNFL and macular data with biometric correlations in Lebanese/Mediterranean children, demonstrating the importance of considering optic nerve head area when interpreting RNFL measurements.10 The consistency of these findings across Mediterranean and Southern European populations supports the validity of our results while highlighting that even among ethnically similar populations, device-specific and methodological differences can produce measurable variations in normative values.

Importantly, Gama et al. (2018) recommended that future normative databases adjust for ONH area due to the positive correlation between pRNFL/GCIPL and ONH area.15 Our study similarly found variations in optic disc area across refractive groups, with myopes presenting smaller disc areas, reinforcing the importance of considering biometric parameters when interpreting pediatric OCT measurements.

A key contribution of our study is the inclusion of subfoveal choroidal thickness (SFCT) measurements, which have not been previously reported in Portuguese pediatric populations.. The finding of significantly thinner SFCT in myopic eyes (298.55 ± 58.52 µm vs 316.58 ± 51.46 µm in emmetropes; p = 0.001) is consistent with a growing body of literature demonstrating that choroidal thinning is associated with myopia and axial elongation in paediatric populations.24–27 Crucially, multivariate analysis confirmed that this association persisted after adjustment for age, sex, and axial length, suggesting that choroidal thinning in myopia reflects a genuine structural correlate of the myopic process rather than a secondary effect of biometric differences between groups. This pattern aligns with previous paediatric studies reporting choroidal thinning in association with refractive error and ocular growth, particularly in myopia.5,24 The observed negative correlation between axial length and choroidal thickness supports the hypothesis that axial elongation is a major driver of choroidal thinning during childhood and adolescence.25

Interestingly, no significant differences were found between myopic and hyperopic participants, despite their opposing refractive states. Similar findings have been reported in some paediatric cohorts, suggesting that both excessive axial elongation (myopia) and shorter axial length (hyperopia) may be associated with choroidal remodeling mechanisms that differ from those observed in emmetropic eyes.26 The thicker choroid observed in emmetropic children may reflect a more balanced biomechanical and vascular environment that favors normal ocular growth regulation.

From a physiological perspective, the choroid plays a critical role in ocular growth modulation by regulating scleral remodeling and retinal signaling pathways. Animal and human studies suggest that choroidal thinning may precede or accompany myopia progression, highlighting its potential role as an early biomarker of refractive development.27 Our findings reinforce the importance of considering refractive status when interpreting pediatric choroidal measurements.

Central foveal thickness did not differ significantly between refractive groups in univariate analyses (p = 0.727), and this finding was confirmed by multivariate regression, where only axial length was independently associated with foveal thickness. This result is consistent with several pediatric OCT studies reporting relative stability of central macular thickness across refractive categories.1,5 The absence of significant differences at the foveal center may be explained by the specialized anatomical structure of the fovea, which is relatively preserved during axial elongation, particularly in eyes with axial length ≤26 mm, as included in this study.28

However, significant differences were detected in macular area and volume parameters, with emmetropic and hyperopic children exhibiting slightly higher values compared to myopes.29 These findings suggest that while central thickness remains stable, peripheral macular regions may be more susceptible to the mechanical stretching associated with axial elongation in myopia. Similar trends have been described in adolescent and young adult populations, indicating that volumetric macular metrics may be more sensitive markers of early structural changes than point thickness measurements alone.30

In univariate analyses, global RNFL thickness was significantly reduced in myopes compared with emmetropes (108.29 vs 114.18 µm; p < 0.001), consistent with prior reports in paediatric populations.31–34 However, the most important finding from the multivariate analysis was that refractive group was no longer a significant independent predictor of RNFL thickness after adjustment for optic disc area and axial length. This finding aligns with the recommendation by Gama et al. (2018)15 to adjust RNFL values for optic nerve head area, and highlights the risk of misinterpreting univariate RNFL differences between refractive groups as direct effects of refractive status when they may instead reflect differences in optic disc anatomy and axial dimensions. Clinically, this underscores the importance of considering optic disc area when interpreting RNFL measurements in myopic children.

Multivariate regression analyses demonstrated that axial length showed stronger and more consistent associations with structural OCT parameters than refractive group classification alone. After adjustment for potential confounders, longer axial length remained independently associated with thinner RNFL thickness and greater foveal thickness, suggesting that ocular elongation may better reflect anatomical retinal variation than categorical refractive status.35 In contrast, choroidal thickness exhibited a distinct pattern, remaining independently associated with age, axial length, and refractive group.36 The persistence of the association between refractive status and choroidal thickness after biometric adjustment suggests that the choroid may be more sensitive to refractive development processes beyond ocular elongation alone.36,37 These findings support the inclusion of biometric covariates, particularly axial length, when establishing pediatric normative OCT databases and highlight subfoveal choroidal thickness as a potentially sensitive structural biomarker of myopic development in school-aged children.

The establishment of population-specific normative OCT data is essential for improving diagnostic accuracy in pediatric ophthalmology. Applying adult or non-European reference databases to Portuguese children may result in misclassification, particularly in the context of myopia assessment and glaucoma suspicion. The normative values presented in this study provide a valuable reference framework for clinicians and may facilitate earlier and more accurate identification of abnormal structural changes. Furthermore, the demonstrated associations between refractive status, axial length, and OCT parameters highlight the importance of integrating biometric and refractive information into OCT interpretation. Longitudinal studies are warranted to determine whether the observed structural differences predict future myopia progression or optic nerve vulnerability.

Several limitations of this study should be acknowledged. First,non-cycloplegic refraction was used for refractive group classification. Non-cycloplegic refraction may result in a myopic shift of approximately 0.25–0.75 D in children, with greater variability in younger age groups, potentially leading to misclassification at the boundaries between refractive categories, particularly given the narrow emmetropic range (±0.50 D) used. This limitation is shared by many large-scale school-based paediatric studies where cycloplegia is logistically or ethically impractical. The same cohort has been previously characterised using non-cycloplegic refraction methodology,18 and findings should be interpreted within this methodological framework. Nonetheless, future studies should employ cycloplegic refraction to provide more accurate refractive group classification, particularly in younger children.

To explore the potential influence of accommodative bias, additional subgroup analyses were performed in participants aged 12 years or older, in whom accommodation-related measurement errors are expected to be less pronounced. The main associations between refractive status and OCT parameters remained generally consistent in this older subgroup, although effect sizes were mostly small to moderate. Nevertheless, some degree of refractive misclassification cannot be excluded and should be considered when interpreting these normative data. Second, the OCT device used in this study (Mocean 3000, SLOOCT) is a less commonly cited platform in the published paediatric OCT literature compared with more widely used systems such as the Cirrus HD-OCT or Heidelberg Spectralis. This limits direct numerical comparison with normative datasets obtained using other devices, and absolute thickness values should be interpreted with this caveat. Formal intraclass correlation coefficient (ICC) validation for the Mocean 3000 was not performed in this study; future studies using this platform should report ICC values to establish device-specific reliability benchmarks. Third, this is a cross-sectional study, precluding causal inference regarding the relationship between refractive development and OCT parameters. Longitudinal studies are needed to assess how these parameters evolve with myopia progression in Portuguese children.